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and Their Association with Smoking and Microvascular Complications in Young Adults with Type 1 Diabetes
Division of Endocrinology and Metabolic Diseases (G.Z., M.M., G.T.), Division of Clinical Chemistry (G.F.), University of Verona Medical School, Verona; and Diabetes Unit (L.Z., G.F., G.T.), Sacro Cuore Hospital of Negrar, Verona, Italy 37024
Address all correspondence and requests for reprints to: Giovanni Targher, M.D., Servizio di Diabetologia, Ospedale Sacro Cuore, via Sempreboni, 5, 37024 Negrar (VR), Italy. E-mail: targher{at}sacrocuore.it; or Giacomo Zoppini, M.D., Division of
Abstract
The purposes of this study were 1) to compare soluble tumor
necrosis factor-
receptors, which are thought to reflect the degree
of TNF- activation, in nondiabetic subjects and type 1 diabetic
patients, and 2) to evaluate the effects of smoking and microvascular
complications on soluble tumor necrosis factor- receptor levels in
type 1 diabetic individuals. Plasma soluble tumor necrosis factor-
receptor levels (R1 and R2) were measured in 50 young type 1 diabetic
patients without clinical macroangiopathy and in a matched group of 20
healthy volunteers. When diabetic patients were grouped according to
smoking and microvascular complication status, the groups of patients
had similar values of age, sex, body mass index, blood pressure,
lipids, creatinine, and glycometabolic control. Nevertheless, soluble
tumor necrosis factor- receptor-R1 levels but not R2 levels, were
markedly elevated (P < 0.05 or less) in
complicated vs. uncomplicated (2.40 ± 0.3
vs. 1.80 ± 0.1 ng/ml) patients and in smokers
vs. nonsmokers (2.66 ± 0.4 vs.
1.76 ± 0.1 ng/ml). In a two-factor ANOVA, both smoking
(P < 0.01) and microvascular complications
(P < 0.05) were independent predictors of soluble
tumor necrosis factor- receptor-R1. Soluble tumor necrosis
factor- receptor levels of diabetic patients who did not smoke or
without complications were similar to those of healthy controls. In
conclusion, smoking and microvascular complications seem to exert an
additive and deleterious impact on TNF- activation, as reflected by
levels of soluble tumor necrosis factor- receptors, in young adults
with type 1 diabetes.
TNF- IS A POTENT proinflammatory
cytokine, primarily involved in the pathogenesis of atherosclerosis
(1, 2, 3). TNF- signals through at least two known
cell-surface receptors, termed TNF-R1 and TNF-R2 (2, 3).
Both of these receptors also exist in soluble forms, apparently derived
by proteolytic cleavage of the extracellular portions of the
cell-surface forms. Although the exact function of soluble tumor
necrosis factor- receptors (sTNF-Rs) remains unknown, it has been
suggested that they represent a buffer system that may prolong the
biological actions of TNF- (2, 3, 4, 5). Compared with
circulating TNF-, sTNF-R levels remain elevated for longer periods
of time and are of more value for monitoring inflammatory responses.
Therefore, sTNF-R levels reflect the degree of TNF- activation more
accurately than the measurement of TNF- itself (2, 3, 4, 5). In addition,
elevated sTNF-R1 levels have recently been found to also be predictive
of the risk of cardiovascular mortality in patients with chronic heart
failure (6).
There is limited and somewhat conflicting information regarding the
effect of the diabetic state per se on the activation of
TNF-, particularly in type 1 diabetic individuals. Plasma levels of
sTNF-Rs have been found to be normal (7) or lower
(8, 9) in type 1 diabetic individuals without
microvascular complications, compared with those in control subjects.
On the contrary, sTNF-R levels have been found to be higher in type 1
diabetic patients with microvascular complications (7, 9).
Although higher plasma TNF- levels have been demonstrated in
nondiabetic smokers vs. nonsmokers (10, 11), to
our knowledge, there is a lack of available data regarding the impact
of chronic smoking on sTNF-R levels in young type 1 diabetic
individuals.
Thus, the main purposes of this study were to compare sTNF-R levels, as
sensitive markers of TNF- activation, in nondiabetic subjects and
type 1 diabetic individuals and to evaluate the effects of chronic
smoking and microvascular complications on sTNF-R levels in young type
1 diabetic individuals without clinical evidence of
macroangiopathy.
Materials and Methods
After written informed consent was obtained, 70 subjects were studied: 20 were nondiabetic and 50 had type 1 diabetes mellitus. The patients were randomly selected among outpatients attending our diabetes clinic after exclusion of: 1) subjects with age > 50 yr; 2) subjects with a poor glycometabolic control; 3) subjects taking any other medications besides insulin; and 4) subjects who had a history of recent acute illness or clinical evidence suggestive of any cardiovascular events or kidney or liver diseases. To exclude the presence of clinical macrovascular complications, a resting electrocardiogram, measurement of ankle brachial pressure index, and carotid ultrasonography were performed in all of the diabetic patients. A total of 20 healthy volunteers (recruited from hospital staff members and relatives) matched for age, sex, body mass index (BMI), blood pressure, lipids, and smoking status formed the control group. Information on smoking status was obtained from all of the participants through a questionnaire. Subjects were categorized into those who had never smoked or quit smoking and those who currently smoked.
Venous blood was drawn in the morning (0800–0830 h) after an overnight fast. Plasma glucose, creatinine, lipids, and other biochemical blood measurements were determined by an automatic calorimetric method (DAX 96; Bayer Corp. Diagnostics, Milan, Italy). Glycosylated hemoglobin A1c (HbA1c) was measured by an HPLC analyzer (Bio-Rad Diamat, Milan, Italy); normal range values in our laboratory were 3.0–5.5%. Determination of plasma sTNF-R1 and sTNF-R2 levels was performed in duplicate with a commercially available ELISA kit (Bender MedSystems Diagnostics, Vienna, Austria) with intra- and interassay coefficients of variations of less than 2.5% and 8.0%, respectively, for both sTNF-Rs. In all patients, 24-h urine collections were obtained to assess albumin excretion rate using a RIA method. Patients were categorized as normoalbuminuric (albumin excretion rate < 20 µg/min), microalbuminuric (20–200 µg/min), and macroalbuminuric (> 200 µg/min). No patients had macroalbuminuria. Presence of retinopathy was diagnosed by a single ophthalmologist after pupillary dilation, according to severity scale proposed by the Early Treatment Diabetic Retinopathy Study (12). The prevalence of microvascular complications was similar to that described in other European populations with comparable age, diabetes duration, glycometabolic control, and smoking status (13).
Statistical analysis
Data are presented as means ± SE. The following statistical tests were conducted: unpaired t test, Pearson’s product-moment correlation, one-way ANOVA, two-factor ANOVA, analysis of covariance, and chi-square test (for categorical variables). To improve skewness and kurtosis of the distributions, plasma levels of sTNF-R1, sTNF-R2, and triglycerides were logarithmically transformed for statistical analyses and then back-transformed to their natural units for presentation in tables and figures. Distributions of all other variables were normal. P values < 0.05 were considered statistically significant.
Results
Table 1
shows the baseline
characteristics of control subjects and type 1 diabetic patients
grouped according to smoking and microvascular complications. All
groups of subjects had similar values of age, sex, BMI, blood pressure,
plasma creatinine, total cholesterol, and HbA1c. The prevalence of
smoking was comparable in complicated vs. uncomplicated
patients. The prevalence of microvascular complications was similar in
smokers vs. nonsmokers. Diabetes duration was significantly
longer in complicated patients, whereas plasma triglycerides were
significantly higher in diabetic smokers. The sTNF-R1 levels were
markedly elevated in complicated vs. uncomplicated patients
and in smokers vs. nonsmokers. These results remained
unchanged after adjustment for age, sex, BMI, diabetes duration, blood
pressure, lipids, and HbA1c (data not shown). The sTNF-R1 levels of
healthy controls, independent of their smoking status, were similar to
those of uncomplicated or nonsmoking diabetic patients but
significantly lower than those of complicated or smoking patients. No
significant differences were found in sTNF-R2 levels among the
groups.
|
, sTNF-R1 levels
increased markedly with the increase in the number of cigarettes smoked
per day in diabetic patients. Similarly, sTNF-R1 levels were
significantly higher in patients with microalbuminuria vs.
normoalbuminuria and in patients with proliferative retinopathy,
compared with those with background retinopathy or with no retinopathy.
Plasma sTNF-R2 did not significantly differ among the groups (data not
shown). Patients with microalbuminuria alone (n = 3) or with
retinopathy alone (n = 13) significantly differed in their
circulating sTNF-R1 levels, compared with patients (n = 7) with
combined microalbuminuria and retinopathy (1.64 ± 0.02
vs. 1.90 ± 0.2 vs. 3.37 ± 1.1 ng/ml;
P = 0.023 for trend).
|
, a two-factor ANOVA
was performed to discriminate separately the adverse effects of smoking
and complication status on sTNF-R1 levels. Cigarette smoking
(P < 0.01) and microvascular complications
(P < 0.05) had an additive and deleterious impact on
sTNF-R1 levels. The adjustment for age, sex, BMI, diabetes duration,
blood pressure, lipids, and HbA1c did not significantly modify these
results (data not shown).
|
Discussion
The main findings of this study are that 1) in type 1 diabetic patients who smoked currently or with microvascular complications, there is a marked increase in sTNF-R1 levels, compared with diabetic nonsmokers or without complications or healthy subjects; 2) uncomplicated diabetic nonsmokers have sTNF-Rs levels similar to those of healthy subjects; and 3) smoking and microvascular complications exert an additive and deleterious effect on sTNF-R1 levels; the effect of smoking is strictly dose dependent, whereas that of the microvascular complications is significantly associated with their degree of severity.
Importantly, in this study we have evaluated the effects of smoking and microvascular complications on sTNF-Rs levels in young adults with type 1 diabetes who were without any clinical evidence of macrovascular complications; the evaluation of patients with such complications would have confounded the interpretation of data. Moreover, the fact that the subgroups of diabetic patients were comparable for several factors known to adversely affect sTNF-Rs levels enhances the validity of our findings.
Based on the present results, therefore, it seems reasonable to
speculate that the observed difference between smokers and nonsmokers
in sTNF-R1 levels was secondary to cigarette smoking and,
theoretically, could be caused by direct effects of nicotine or other
agents contained in tobacco smoke. It is known that smoking may
directly influence both the production of cytokines and acute-phase
proteins and the antioxidant defenses by exerting its chronic
inflammatory stimulus on the macrophage-monocyte system (1, 10, 11). The biological relevance of TNF- activation to the
pathogenesis and progression of diabetic microvascular complications
remains uncertain. It is possible that TNF- activation is simply an
epiphenomenon of some aspect of the process causing the diabetic
complications. However, recent studies support the possibility that
TNF- activation may, directly or indirectly, contribute to the
pathogenesis and development of microvascular complications (14, 15). This possibility lends itself to testing using
interventions to influence TNF- secretion and actions. The
mechanisms that trigger the activation of TNF- in type 1 diabetes
are not fully understood, but it is likely that local hypoxia, induced
by capillary occlusion, and high levels of advanced glycosylation
end-products, which are associated with the development of diabetic
complications, may induce TNF- activation (14, 15, 16). Our
finding of a strong relationship between sTNF-R1 and fibrinogen
concentrations, which was significant even after adjustment for smoking
and complication status, probably reflects the fact that TNF-, along
with other cytokines, is a primary stimulant for the hepatic production
of acute-phase proteins, such as fibrinogen and C-reactive protein.
This suggests the possibility of an additional pathophysiological
mechanism by which TNF- activation may play a detrimental role in
the development of diabetic complications.
The reason why the adverse effects of smoking and complication status are predominantly observed on sTNF-R1 levels is not well understood. The present finding, however, is in accord with that also reported by others (7, 9). The sTNF-R1 levels have been proposed to play a primary role in the inflammatory processes associated with atherogenesis (2, 3). Furthermore, in a recent study, elevated levels of sTNF-R1, but not of sTNF-R2, independently predicted the risk of cardiovascular mortality in patients with chronic heart failure (6).
Overall, therefore, the evidence from this and other studies (7) suggests that type 1 diabetic smokers or with microvascular complications have markedly elevated sTNF-R1 levels, compared with diabetic nonsmokers or without complications or healthy subjects. Moreover, smoking itself appears to be a major lifestyle determinant of sTNF-R1 levels in both normal subjects (10, 11) and type 1 diabetic individuals. In line with previous studies (7, 8, 9), sTNF-Rs levels did not significantly correlate to HbA1c or lipids in type 1 diabetic patients.
Although the evidence of a relationship between smoking and diabetic complications is still conflicting, a number of studies have reported a strong relationship between smoking and the progression of microvascular diseases, thus supporting the possibility that quitting smoking would be effective in the reducing the incidence of complications (17, 18).
In conclusion, although this study is cross-sectional and cannot
therefore prove a direct cause-effect relationship, the present results
suggest that chronic smoking and microvascular complications can exert
an additive and deleterious impact on TNF- activation, as reflected
by levels of sTNF-Rs, in young adults with type 1 diabetes.
Footnotes
Abbreviations: BMI, Body mass index; HbA1c, hemoglobin A1c;
sTNF-Rs, soluble tumor necrosis factor- receptors.
Received January 26, 2001.
Accepted April 25, 2001.
References
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activity by TNF receptors in patients with proliferative diabetic
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